I. Field of the Invention
The present invention relates to a novel ALS3 promoter which generally drives constitutive and generally non-tissue-preferred expression of operably linked foreign genes in transformed plants. In particular, this invention is directed to DNA constructs in which a Brassica ALS3 promoter is operably linked to a foreign structural gene, and to using the DNA construct to produce, in a transformed plant, a protein which is encoded by the structural gene. The Brassica ALS3 promoter is used to direct expression of agronomically important genes and selectable marker genes.
II. Background
Acetolactate synthase (ALS), which is also known as acetohydroxy acid synthase (AHAS), catalyses the first step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine. It has also been shown to be the site of action of sulfonylurea and imidazolinone based herbicides. See, for example, Chaleff, R. S. and C. J. Mauvais, Science 224:1443 (1984) and Shaner et al., Plant Physiol. 76:545 (1984). A number of different ALS genes from Brassica napus have been cloned and characterized. See, for example Wiersma et al., Mol. Gen. Genetics 219:413 (1989) and Rutledge et al., loc. cit. 229:31 (1991).
Rutledge et al. (1991) reported that the B. napus rapeseed cultivar Topas contains an ALS multigene family comprised of five genes. DNA sequence analysis of the structural genes revealed that the ALS1 and ALS3 genes shared extensive sequence homology. In contrast, the ALS2 gene has diverged significantly from the ALS1 and ALS3 genes and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding region. The ALS2 gene therefore may encode a polypeptide with a distinct function from that of ALS1 and ALS3. The ALS4 and ALS5 genes have interrupted coding regions and therefore may be defective.
Experiments conducted with the promoter of the Arabidopsis thaliana ALS gene revealed that the A. thaliana ALS promoter is significantly less effective in driving gene expression than the CaMV 35S promoter. Odell et al., Plant Physiol. 94(4):1647-1654 (1990) replaced the A. thaliana ALS promoter with the CaMV 35S promoter and observed a 25-fold increase in the level of ALS mRNA accompanied by a 2-fold increase in ALS enzyme level and a 3-fold increase in sulfonylurea tolerance. These observations indicate that the ALS gene is regulated post-transcriptionally and that the A. thaliana ALS promoter is significantly less effective in driving gene expression than the CaMV 35S promoter.
The number of isolated and characterized constitutive generally non-tissue-preferred plant promoters available for expression of foreign proteins in transgenic plants is very limited. Well known examples of promoters with constitutive and tissue generated expression patterns include those associated with the CaMV 35S, Agrobacterium nopaline synthase, and maize ubiquitin genes. See Odell et al., Plant Mol. Biol. 10(3):263-272 (1988), Herrera-Estrella et al., Nature 303:209-213 (1983) and Fox et al., Va. J. Sci. 43(2):287 (1992).
There is a critical need for a broader repertoire of strong constitutive and generally non-tissue-preferred plant promoters. A broader array of constitutive and generally non-tissue-preferred plant promoters that are expressed at high levels, that is, that drive expression of operably linked genes at a level comparable to the CaMV 35S promoter, would allow the genetic engineer to analyze the relative strengths of the available promoters and select promoters that provide the required level of expression of foreign genes in transformed plants. A selected promoter might provide optimum levels of expression for the first gene but may be either too strong or too weak for use in driving the expression of a second gene. Consequently, additional constitutive and tissue general promoters are needed to optimize foreign gene expression in plants.
There is also a need for additional strong constitutive and generally non-tissue preferred promoters for construction of plants transformed with multiple foreign genes. Numerous difficulties have arisen when two or more different genes are introduced into a plant wherein each of the genes are operably linked to the same or similar promoters. Some of these difficulties include (1) gene inactivation; (2) recombination as a result of pairing along homologous regions within the nucleotide sequence of the promoter leading to cross-over events and loss of the intervening region prior, or subsequent to, integration; and (3) competition among different copies of the same promoter region for binding of promoter-specific transcription factors or other regulatory DNA-binding proteins. A need therefore exists for a broader repertoire of strongly constitutive and tissue general promoters to be used for expression of foreign genes in transformed plants.